The present invention relates to sequences coding for plasma proteins in general and, more specifically, to a DNA sequences which codes for a protein having substantially the same structure and/or activity of human protein C.
Protein C is a zymogen, or precursor, of a serine protease which plays an important role in the regulation of blood coagulation and generation of fibrinolytic activity in vivo. It is synthesized in the liver as a single-chain polypeptide which undergoes considerable processing to give rise to a two-chain molecule comprising heavy (Mr=40,000) and light (Mr=21,000) chains held together by disulphide bonds. The circulating two-chain intermediate is converted to the biologically active form of the molecule, known as xe2x80x9cactivated protein Cxe2x80x9d (APC), by the thrombin-mediated cleavage of a 12-residue peptide from the amino-terminus of the heavy chain. The cleavage reaction is augmented in vivo by thrombomodulin, an endothelia cell cofactor (Esmon and Owen, Proc. Natl. Acad. Sci. USA 78: 2249-2252, 1981).
Protein C is a vitamin K-dependent glycoprotein which contains approximately eleven residues of gammacarboxyglutamic acid (gla) and one equivalent of betahydroxyaspartic acid which are formed by post-translational modifications of glutamic acid and aspartic acid residues, respectively. The post-translational formation of specific gamma-carboxyglutamic acid residues in protein C requires vitamin K. These unusual amino acid residues bind to calcium ions and are believed to be responsible for the interaction of the protein with phospholipid, which is required for the anticoagulant activity of protein C.
In contrast to the coagulation-promoting action of other vitamin K-dependent plasma proteins, such as factor VII, factor IX, and factor X, activated protein C acts as regulator of the coagulation process through the inactivation of factor Va and factor VIIa by limited proteolysis. The inactivation of factors Va and VIIIa by protein C is dependent upon the presence of acidic phospholipids and calcium ions. Protein S has been reported to regulate this activity by accelerating the APC-catalyzed proteolysis of factor Va (Walker, J. Biol. Chem. 255:5521-5524, 1980).
Protein C has also been implicated in the action of plasminogen activator (Kisiel and Fujikawa, Behring Inst. Mitt. 73:29-42, 1983). Infusion of bovine APC into dogs results in increased plasminogen activator activity (Comp and Esmon, J. Clin. Invest. 68: 1221-1228, 1981). Recent studies (Sakata et al., Proc. Natl. Acad, Sci. USA 82: 1121-1125, 1985) have shown that addition of APC to cultured endothelia cells leads to a rapid, dose-dependent increase in fibrinolytic activity in the conditioned media, reflecting increases in the activity in both urokinase-related and tissue-type plasminogen activators by the cells. APC treatment also results in a dose-dependent decrease in antiactivator activity.
Inherited protein C deficiency is associated with recurrent thrombotic disease (Broekmans et al., New Eng. J. Med. 309: 340-344, 1983; and Seligsohn et al., New Eng. J. Med. 310: 559-562, 1984) and may result from genetic disorder or from trauma, such as liver disease or surgery. This condition is generally treated with oral anti-coagulants. Beneficial effects have also been obtained through the infusion of protein C-containing normal plasma (see Gardiner and Griffin in Prog. in Hematology, ed. Brown, Grune and Stratton, NY, 13: 265-278). In addition, some investigation have discovered that the anti-coagulant activity of protein C is useful in treating thrombotic disorders, such as venous thrombosis (WO 85/00521). In some parts of the world, it is estimated that approximately 1 in 16,000 individuals exhibit protein C deficiency. Further, a total deficiency in protein C is fatal in newborns.
While natural protein C may be purified from clotting factor concentrates (Marlar et al., Blood 59: 1067-1072) or from plasma (Kisiel, ibid), it is a complex and expensive process, in part due to the limited availability of the starting material and the low concentration of protein C in plasma. Furthermore, the therapeutic use of products derived from human blood carries the risk of disease transmission by, for example, hepatitis virus, cytomegalovirus, or the causative agent of acquired immune deficiency syndrome (AIDS). In view of protein C""s clinical applicability in the treatment of thrombotic disorders, the production of useful quantities of protein C and activated protein C is clearly invaluable.
Briefly stated, the present invention discloses a DNA sequence which codes for a protein having substantially the same biological activity as human protein C.
In addition, the present invention discloses a recombinant plasmid or bacteriophage transfer vector comprising a cDNA sequence comprising the protein C gene cDNA sequence. The amino acid and DNA sequences of this cDNA coding for human protein C are also disclosed.
Other aspects of the invention will become evident upon reference to the detailed description and attached drawings.